Project Summary/Abstract Many of the flavoring chemicals in e-cigarettes present a class of chemicals unique to e-cigarettes, potentially causing distinct adverse health effects. Several e-liquid flavoring compounds are ?,?-unsaturated aldehydes, a class of chemicals with known adverse effects on respiratory immune function. Among those flavoring compounds is cinnamaldehyde (CA), an ?,?-unsaturated aldehyde often contained in popular cinnamon or spicy flavored e-liquids and with known immune modulating activities. However, potential effects of CA on respiratory immune responses present a critical knowledge gap, which will be the focus of this application. We will use tightly linked mechanistic in vitro and human in vivo studies to determine adverse effects of CA on respiratory innate immune functions, with specific focus on two components: 1) the mucociliary component consisting of ciliated epithelial cells lining the airways and 2) the cellular component consisting of resident and infiltrating leukocytes, such as macrophages (Macs). Our data demonstrate that CA-containing e-liquids greatly affect ciliary beating and respiratory immune cell function at doses that do not cause overt cytotoxicity. These effects were associated with modified mitochondrial respiration and could be inhibited by thiol reducing reagents. Thus, based on existing knowledge and our own data we hypothesize that CA-containing e-liquids suppress innate mucosal immune function by CA-induced inhibition of mitochondrial respiration and thiol modification of cellular proteins. We will test this hypothesis in two specific aims: SA1 will determine CA- induced effects on epithelial ciliary function and mucociliary clearance (MCC) and identify the mechanisms mediating these responses. To achieve this aim we will expose well-differentiated human bronchial epithelial cells (HBECs) to CA-containing e-liquids, assess changes in ciliary beating, and determine the role of mitochondrial respiration and thiol modification in these responses. To translate these findings into humans in vivo, we propose to have healthy adult volunteers undergo controlled inhalation of Technetium-99m sulfur colloid (Tc99m-SC) particles after inhalation of CA-containing e-cigarettes, followed by tracking the egress of the radiolabeled particles as a measure of MCC using gamma scintigraphy. SA2 will determine CA-induced modulation of Macs and the mechanisms mediating these responses. To achieve this aim we will stimulate human Macs with CA-containing e-liquids ex vivo and examine changes in immune function, and determine the role of thiol modification and mitochondrial respiration in these responses. Macs obtained through induced sputum (IS) from human subjects undergoing controlled vaping exposure to CA-containing e-liquids will be used to translate the mechanistic findings obtained in Macs ex vivo into humans in vivo. Data derived from these highly integrated translational studies will yield important mechanistic information on a popular e- cigarette flavoring agent with potential implications for a larger group of chemical flavoring agents, thus addressing a clinical knowledge gap related to the potential health effects of flavored e-cigarette use.